In a living cell, from the moment a primary RNA transcript is complete to the actual expression of the protein encoded by the transcript, multiple cellular events and mechanisms occur, including pre-RNA splicing, RNA editing, shuttling of the mRNA between the nucleus and the cytoplasm, and ensuring the stability and translational control of the trafficked mRNAs. Each of these events provides opportunities for the cell to regulate gene expression at the RNA level.
Recent studies have revealed that RNA binding proteins (RBPs) are crucial functional components of the molecular “machinery” involved in each of these key post-transcriptional events (Maquat, L. E. et al., Cell, 104, 173-6 (2001)). Disruption of these RBPs, also known as “cellular integrators,” has been implicated in the pathogenesis of epilepsy (Musunuru, K., et al., Annu. Rev. Neurosci., 24, 239-62 (2001)), rheumatism (Fritsch, R. et al., J. Immunol., 169, 1068-76 (2002)), cancer, motor neuron disease (Pellizzoni, L., et al., Cell, 95, 615-24 (1998)), and mental retardation (Turner, G., et al., Am. J. Med. Genet., 64, 196-7 (1996)). The current lack of available therapeutic tools exists, in part, because so few in vivo RNA-protein complexes have been identified and characterized. Therefore, identification of specific RNAs and the proteins with which they form ribonucleoprotein (RNP) complexes will enable the development of therapeutic tools, such as the regulation of gene expression. This will in turn enable the use of biological manipulation of gene expression in the laboratory and develop its use as a therapeutic tool for cellular processes that are not currently understood.
Several methods have previously been used to understand RNA-protein binding activity. These methods include filter binding assays, UV cross-linking assays, and gel shift assays. Gel shift assays, for example, are commonly used to confirm RNA binding activity by showing that RNA migrates at a higher molecular weight after incubation with protein, suggestive of interaction between the RNA and protein. Subsequently, a supershift assay consisting of the exposure of the RNA-protein complex, or ribonucleoprotein (RNP), to an antibody generated against the alleged RBP confirms the presence of the protein in the RNP complex when this RNP-Ab complex migrates at a rate of an even higher molecular weight species within the electrophoretic gel. The utility and applicability of the results obtained using these classical methods is limited with respect to obtaining a detailed understanding of RNA binding activity, since the methods reveal only which binding interactions occur in vitro. Other less conventional techniques have been devised to address this concern (Tenenbaum, S. A., et al., PNAS, 97, 14085-90 (2000), Brodsky, A. S., et al., Molecular & Cellular Proteomics, 1.12, 922-9 (2002)), but these methods still rely on in vitro techniques in their methodology, as is the case, for example, with the use of immunoprecipitation (IP) to assess RNA targets of embryonic lethal abnormal visual system (ELAV)-like neuronal RNA-binding protein “HuB” via cDNA arrays (Tenenbaum, S. A., et al., PNAS, 97, 14085-90 (2000)). To truly understand the dynamics of RNA-protein interactions, it is first necessary to possess the ability to identify the interactions in vivo. In an attempt to identify in vitro interactions, Miyashiro et al. have developed the APRA (antibody-positioned RNA amplification) methodology, which identifies RNA cargoes that complex in vivo with the antibody's target protein (Miyashiro, K. Y. et al., Neuron, 37, 417-31 (2003)). However, this technique also suffers from several deficiencies, including the requirement that the identity of the RNA binding protein must first be known, and unknown proteins cannot therefore be identified with the technique.
Each of these procedures permits the characterization of RNA cargoes that bind to a particular RBP. However, in order to characterize the RBPs that bind to any particular RNA, the existing methodologies are cumbersome and complex, they require a significant amount of time, they require large amounts of starting material, and they lead to many false positives. Additionally, all of the existing assays that attempt such characterization utilize in vitro methodologies. What is needed is a methodology that provides for the identification of proteins that interact in vivo with a target mRNA. Therefore, there exists a long felt need to provide a way to identify proteins that interact with a pre-selected RNA in vivo. The present invention addresses and meets this need.